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Xanthomonas oryzae pv. oryzae type III effector XopN targets OsVOZ2 and a putative thiamine synthase as a virulence factor in rice.

Cheong H, Kim CY, Jeon JS, Lee BM, Sun Moon J, Hwang I - PLoS ONE (2013)

Bottom Line: In contrast, the xopN KXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85.The wild-type KXO85 and xopN KXO85 mutant were significantly less virulent in the mutant rice line.These results indicate that XopNKXO85 and OsVOZ2 play important roles both individually and together for Xoo virulence in rice.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

ABSTRACT
Xanthomonasoryzae pv. oryzae (Xoo) is spread systemically through the xylem tissue and causes bacterial blight in rice. We evaluated the roles of Xanthomonas outer proteins (Xop) in the Xoo strain KXO85 in a Japonica-type rice cultivar, Dongjin. Five xop gene knockout mutants (xopQ KXO85 , xopX KXO85 , xopP1 KXO85 , xopP2 KXO85 , and xopN KXO85 ) were generated by EZ-Tn5 mutagenesis, and their virulence was assessed in 3-month-old rice leaves. Among these mutants, the xopN KXO85 mutant appeared to be less virulent than the wild-type KXO85; however, the difference was not statistically significant. In contrast, the xopN KXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85. These observations indicate that the roles of Xop in Xoo virulence are dependent on leaf stage. We chose the xopN gene for further characterization because the xopN KXO85 mutant showed the greatest influence on virulence. We confirmed that XopNKXO85 is translocated into rice cells, and its gene expression is positively regulated by HrpX. Two rice proteins, OsVOZ2 and a putative thiamine synthase (OsXNP), were identified as targets of XopNKXO85 by yeast two-hybrid screening. Interactions between XopNKXO85 and OsVOZ2 and OsXNP were further confirmed in planta by bimolecular fluorescence complementation and in vivo pull-down assays. To investigate the roles of OsVOZ2 in interactions between rice and Xoo, we evaluated the virulence of the wild-type KXO85 and xopN KXO85 mutant in the OsVOZ2 mutant line PFG_3A-07565 of Dongjin. The wild-type KXO85 and xopN KXO85 mutant were significantly less virulent in the mutant rice line. These results indicate that XopNKXO85 and OsVOZ2 play important roles both individually and together for Xoo virulence in rice.

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Localization of XopNKXO85, OsVOZ2, and OsXNP in plant cells.Subcellular localization of the XopNKXO85-GFP, OsVOZ2-GFP, and OsXNP-GFP fusion proteins in maize mesophyll cells. OsABF1-RFP was used as a nuclear marker. GFP (green) fluorescence was merged with RFP (red) fluorescence. Bars = 10 µm.
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pone-0073346-g003: Localization of XopNKXO85, OsVOZ2, and OsXNP in plant cells.Subcellular localization of the XopNKXO85-GFP, OsVOZ2-GFP, and OsXNP-GFP fusion proteins in maize mesophyll cells. OsABF1-RFP was used as a nuclear marker. GFP (green) fluorescence was merged with RFP (red) fluorescence. Bars = 10 µm.

Mentions: To determine their subcellular localizations, XopNKXO85, OsVOZ2, and OSXNP were tagged with GFP at their C-termini in p2GWF7-XopN, p2GWF7-OsVOZ2, and p2GWF7-OsXNP, respectively (Table S3). In transient expression assays using maize mesophyll protoplasts, GFP signals from XopN-GFP and OsXNP-GFP were mostly detected in the cytoplasm, whereas those from OsVOZ2-GFP were detected in both the cytoplasm and the nucleus compared to the nuclear marker OsABF1-RFP (Figure 3). These data indicate that XopNKXO85 and OsXNP are localized in the cytoplasm, whereas OsVOZ2 is localized in a nuclear and cytoplasm (Figure 3).


Xanthomonas oryzae pv. oryzae type III effector XopN targets OsVOZ2 and a putative thiamine synthase as a virulence factor in rice.

Cheong H, Kim CY, Jeon JS, Lee BM, Sun Moon J, Hwang I - PLoS ONE (2013)

Localization of XopNKXO85, OsVOZ2, and OsXNP in plant cells.Subcellular localization of the XopNKXO85-GFP, OsVOZ2-GFP, and OsXNP-GFP fusion proteins in maize mesophyll cells. OsABF1-RFP was used as a nuclear marker. GFP (green) fluorescence was merged with RFP (red) fluorescence. Bars = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760903&req=5

pone-0073346-g003: Localization of XopNKXO85, OsVOZ2, and OsXNP in plant cells.Subcellular localization of the XopNKXO85-GFP, OsVOZ2-GFP, and OsXNP-GFP fusion proteins in maize mesophyll cells. OsABF1-RFP was used as a nuclear marker. GFP (green) fluorescence was merged with RFP (red) fluorescence. Bars = 10 µm.
Mentions: To determine their subcellular localizations, XopNKXO85, OsVOZ2, and OSXNP were tagged with GFP at their C-termini in p2GWF7-XopN, p2GWF7-OsVOZ2, and p2GWF7-OsXNP, respectively (Table S3). In transient expression assays using maize mesophyll protoplasts, GFP signals from XopN-GFP and OsXNP-GFP were mostly detected in the cytoplasm, whereas those from OsVOZ2-GFP were detected in both the cytoplasm and the nucleus compared to the nuclear marker OsABF1-RFP (Figure 3). These data indicate that XopNKXO85 and OsXNP are localized in the cytoplasm, whereas OsVOZ2 is localized in a nuclear and cytoplasm (Figure 3).

Bottom Line: In contrast, the xopN KXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85.The wild-type KXO85 and xopN KXO85 mutant were significantly less virulent in the mutant rice line.These results indicate that XopNKXO85 and OsVOZ2 play important roles both individually and together for Xoo virulence in rice.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

ABSTRACT
Xanthomonasoryzae pv. oryzae (Xoo) is spread systemically through the xylem tissue and causes bacterial blight in rice. We evaluated the roles of Xanthomonas outer proteins (Xop) in the Xoo strain KXO85 in a Japonica-type rice cultivar, Dongjin. Five xop gene knockout mutants (xopQ KXO85 , xopX KXO85 , xopP1 KXO85 , xopP2 KXO85 , and xopN KXO85 ) were generated by EZ-Tn5 mutagenesis, and their virulence was assessed in 3-month-old rice leaves. Among these mutants, the xopN KXO85 mutant appeared to be less virulent than the wild-type KXO85; however, the difference was not statistically significant. In contrast, the xopN KXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85. These observations indicate that the roles of Xop in Xoo virulence are dependent on leaf stage. We chose the xopN gene for further characterization because the xopN KXO85 mutant showed the greatest influence on virulence. We confirmed that XopNKXO85 is translocated into rice cells, and its gene expression is positively regulated by HrpX. Two rice proteins, OsVOZ2 and a putative thiamine synthase (OsXNP), were identified as targets of XopNKXO85 by yeast two-hybrid screening. Interactions between XopNKXO85 and OsVOZ2 and OsXNP were further confirmed in planta by bimolecular fluorescence complementation and in vivo pull-down assays. To investigate the roles of OsVOZ2 in interactions between rice and Xoo, we evaluated the virulence of the wild-type KXO85 and xopN KXO85 mutant in the OsVOZ2 mutant line PFG_3A-07565 of Dongjin. The wild-type KXO85 and xopN KXO85 mutant were significantly less virulent in the mutant rice line. These results indicate that XopNKXO85 and OsVOZ2 play important roles both individually and together for Xoo virulence in rice.

Show MeSH
Related in: MedlinePlus